Bioprosthetic heart valves (BHVs) derived from glutaraldehyde-crosslinked porcine aortic valves are used annually in thousands of heart valve replacement surgeries. These devices often fail clinically due degeneration and pathologic calcification. Understanding degenerative failure of BHVs in the absence of calcification is rarely addressed. In the previous grant period, we showed that valvular glycosaminoglycans (GAGs) are lost during tissue fixation and after implantation. GAG-degrading enzymes either present in the valve tissue or infiltrated after in vivo implantation are a major cause of GAG degeneration. Loss of GAGs from BHVs leads to decreased tissue flexural rigidity, loss of hysteresis, and collagen structural deterioration. Maintaining the structural integrity of the extracellular matrix (ECM) in the processed tissues is essential for a durable BHV. We found that chemical fixatives alone are only partially effective in preventing GAG loss from BHVs. Our recent results show that addition of neomycin, an inhibitor of GAG-degrading enzymes, in combination with chemical GAG fixation by carbodiimide crosslinking prior to routine glutaraldehyde (GLUT) crosslinking leads to significantly better stabilization of valvular GAGs. The overall aim of this project is extend the durability of BHVs well beyond 20 years. We are proposing to study BHV durability for up to 800 million cycles (25 years of valve functional life). Such long-term fatigue damage study is unprecedented in the BHV field. Thus, we will test the following hypotheses.1) BHVs with improved extracellular matrix stabilization and ethanol pretreatment to prevent calcification will resist degeneration in vitro during extended flexural fatigue and after in vivo implantation. Our novel neomycin-based crosslinking procedure will be combined with clinically used ethanol anti-calcification pretreatment. a) GAG stability will be tested in vitro during storage and cyclic fatigue up to 800 million cycles (more than 25 years of functional life) and b) in vivo in a rat subdermal-implantation model. 2) BHVs with improved extracellular matrix stabilization and ethanol pretreatment for preventing calcification will have improved biomechanical function and enhanced long-term durability. The role of native GAGs in preserving the biomechanical performance of GLUT-crosslinked porcine aortic valve cusps will be studied in two major deformation modes associated with valve function: planar biaxial tension and flexure in presence or absence of GAGs. b) Cuspal biomechanical function during in vitro cyclic fatigue up to 800 million cycles (25 years of functional life) will be studied. 3) BHVs with improved extracellular matrix stabilization and ethanol pretreatment for preventing calcification will be endowed with improved biological durability. Degeneration and calcification of BHVs with GAG-targeted chemistry/GLUT/Ethanol will be compared with clinically used ethanol pretreated GLUT-fixed BHVs in a sheep mitral valve-replacement model. PUBLIC HEALTH RELEVANCE: About 175,000 patients need heart valve replacements due to damaged or dysfunctional valves. Bioprosthetic heart valves derived from chemically fixed pig heart valves are used frequently for this purpose. The majority of these valves fail after 5-15 years due to degeneration and calcification and need replacements again. This grant proposal is investigating new chemical fixatives that would improve functional life-time of these valves so that the valve could outlast the patient.